The purpose of the present study is to determine the mechanisms which direct the cell-specific expression of renin to renal juxtaglomerular cells. The recent development of renin secreting cells (As4.1 cells), produced by targeted tumorigenesis in transgenic animals, provides for the first time a cell culture system in which to carry out these experiments. Previous studies of human renin gene (hREN) expression in transgenic animals indicated that a genomic fragment containing 0.9 kb 5'-flanking DNA, all coding and intervening sequences, plus 0.4 kb 3'-flanking DNA was sufficient and necessary for cell-specific expression at physiological levels. However, the corresponding hREN 5'-flanking DNA sequences linked to a luciferase reporter genes showed very low level activity in both As4.1 cells, and other renin expressing cells. Further analysis of the transgenic mice described above indicated that hREN expression may not be appropriately cell-specific. A sequence from the mouse REN1c 5'-flanking DNA (-4.1 to -2.6 kb) which exhibited the properties of a transcriptional enhancer stimulate hREN promoter activity in both mouse As4.1 cells, and several human renin expressing cell lines. These observations suggested that a similar enhancer element might reside in more distal regions of the hREN 5'-flanking DNA. Since existing hREN genomic clones only contain sequences to approximately -3 kb, a P1 genomic clone was isolated that contains approximately 75 kb spanning hREN. Southern blot analysis revealed a sequence contained with -15 kb of the 5'-flanking DNA which hybridized the mouse enhancer probe. The present study will use a combination of transfection experiments and transgenic mice to study the sequences which direct the cell-specific expression of the human renin gene at physiological levels. Transfection of renin-luciferase hybrid genes into As4.1 cells will be used to determine the activity of the putative enhancer. Further studies will delimit the enhancer and promoter regions required for this activity. Identification of transcription factor binding sites will further define the sequences which direct hREN expression and the mechanisms involved. Studies using fusion genes in which hREN genomic sequences are replaced with the corresponding cDNA will examine the role of intronic sequences. Comparisons between the activities of constructs expressing renin (either as genomic sequences or fusion genes) or a heterologous reporter will examine the role of renin structural sequences. Transgenic mice will be made to determine whether the sequences identified in transfection experiments are necessary and sufficient to direct cell-specific renin gene expression at physiological levels in vivo. Understanding the mechanisms which direct renin gene expression to the renal juxtaglomerular cells may reveal genetic and molecular bases for defects in both essential and high renin hypertension, and for the paradoxical increases in renin secretion that occurs during salt feeding of a number of strains of hypertensive rats. The establishment of transgenic mice in which human renin gene expression more closely resembles the physiological pattern of the endogenous gene will provide better models to study the role of the renin-angiotensin system in the pathogenesis of hypertension.